In recent years, interest in the characterization, biogenesis and function of extracellular vesicles (EVs) has increased immensely. These membrane-derived vesicles play vital roles in a plethora of processes in several biological systems.
Currently, no proteins are known to be constitutively sorted into vesicles independently of the subcellular origin or the activation status of the producing cells. This lack of invariant housekeeping markers hampers the quantitative analysis of the vesicles.
Although the EV phenotype is particularly important in the determination of cellular and subcellular origin, it can in combination with a protein cargo analysis additionally provide clues about the functionality of the EVs. Further refinements of existing methods will not only contribute to broadening our understanding of the biological role of the EVs, but are also likely to accelerate the implementation of EVs as biomarkers in clinical diagnostics and as therapeutic agents.
Several methods exist to characterize the protein composition of EVs related to either a surface marker phenotype or the proteins present in the EV cargo, as reviewed by Revenfeld et al.
Detection and molecular profiling of EVs is technically challenging and often requires extensive sample purification and labelling. Previously, researchers from Aalborg University Hospital developed and described a high-throughput approach for phenotyping EVs. This approach, termed the “EV Array,” is based on a protein microarray platform. Antibodies are printed onto coated glass slides, which enable the capturing of EVs by their surface or surface-associated proteins. Afterwards, profiling of the EVs is performed by detection with selected biotinylated antibodies for exosomes, for example anti-CD9, -CD63 and -CD81. Here the researchers demonstrate an extended microarray approach that offers improved profiling capabilities and provides an easy and efficient way to detect EVs as well as selected subpopulations. Therefore, this method presents an avenue towards scientific, diagnostic and prognostic applications.
Summary of the phenotyping of the exosomal population (positive for CD9, CD63 and/or CD81) in plasma from 5 selected, healthy donors. The exosomes were profiled using an EV Array printed with either 21 (Print 1), 33 (Print 2), 50 (Print 3) or 60 (Print 4) different capturing antibodies. The relative fluorescence intensity was log2 transformed prior to the visualization presented as a heat map. Black indicates no signal and green corresponds to maximum signal as indicated by the color-coded bar.